CM-DIL和DAPI标记的骨髓间充质干细胞商青青;李凯;周建业;胡盛寿【摘要】背景：在有关“细胞移植”的实验研究中，细胞标记技术被广泛应用。
CM-DIL与DAPI是细胞标记实验中常用的荧光染料，目前两者的对比研究报道较少。
<br> 目的：从体外实验与体内实验两方面，探讨两种荧光染料CM-DIL与DAPI在标记大鼠骨髓间充质干细胞方面的差异。
<br> 方法：用贴壁培养法获取、培养、扩增大鼠骨髓间充质干细胞，分别用CM-DIL与DAPI进行标记，锥虫蓝计数检测骨髓间充质干细胞的活力；MTS法检测骨髓间充质干细胞的增殖能力并绘制增殖曲线；倒置相差荧光显微镜下动态观察标记后1，2，3代骨髓间充质干细胞的荧光衰减情况。
结扎SD大鼠冠状动脉前降支致心肌梗死。
1周后于心肌梗死边缘处注射CM-DIL与DAPI标记的细胞，细胞移植后3 d取材观察骨髓间充质干细胞的分布情况。
<br> 结果与结论：体外实验中，CM-DIL与DAPI标记的骨髓间充质干细胞两者的早期增殖能力均低于对照组；两种染料标记后的第1代细胞的荧光阳性率均为100%，但DAPI标记后的第3代细胞的荧光强度明显减弱。
体内实验中，CM-DIL组与DAPI组心肌组织冰冻与石蜡切片均检测到集中分布的荧光；CM-DIL组冰冻切片红色荧光比DAPI组的蓝色荧光边界清晰并且背景低；CM-DIL组还可以通过含细胞核染色的封片剂封片排除荧光假阳性。
可见，CM-DIL染料比DAPI更适合对骨髓间充质干细胞进行体内示踪。
%BACKGROUND:cellmarker technology has been widely applied in many studies concerning celltransplantation. Chlormethylbenzamido-1,1-dioctadecyl-3,3,3’3’-tetramethylin-docarbocyamine (CM-DIL) and 4’,6-diamidino-2-phenylindole (DAPI) are commonly used for labeling cells. To our knowledge, there are few reports on comparing the two fluorescentdyes. <br> OBJECTIVE:To compare the effects of CM-DIL and DAPI on labeling bone marrow mesenchymal stem cells in vitro and in vivo. <br> METHODS:Isolation and expansion of bone marrow-derived mesenchymal stem cells were performed according to attachment culture. The cells were labeled by CM-DIL and DAPI, respectively. cellviability was assessed via trypan blue exclusion assay. Growth curves of bone marrow-derived mesenchymal stem cells were depicted using MTS assay. The reduction of fluorescent intensity was observed under an inverted fluorescent contrast phase microscope from passage 1 to passage 3 after labeling. Myocardial infarction was induced by left anterior artery ligation in Sprague-Dawley rats. One week later, bone marrow-derived mesenchymal stem cells labeled by CM-DIL or DAPI were injected randomly into the border area of infarct myocardium. After 3 days, transplanted celldistribution was examined under the fluorescent microscope through paraffin sections and frozen sections respectively. <br> RESULTS AND CONCLUSION:In vitro, bone marrow-derived mesenchymal stem cells labeled by both CM-DIL and DAPI showed decreased cellproliferation during the early period;the percentage of fluorescent-positive cells was approximately 100%in the two groups;however, the fluorescent intensity was significantly reduced from passage 1 to passage 3 in bone marrow-derived mesenchymal stem cells labeled by DAPI. In vivo, the transplanted cells were detected in a concentrated way both on the paraffin sections and frozen ones;the background color of frozen sections was lower in the CM-DIL group than in the DAPI group;false positive results of fluorescentexpression could be eliminated in the CM-DIL group by using fluorescent mounting medium with the fluorescence of DNA staining. These data indicates that CM-DIL is more appropriate to in vivo tracing cells than DAPI.【期刊名称】《中国组织工程研究》【年(卷),期】2013(000)045【总页数】6页(P7855-7860)【关键词】干细胞;骨髓干细胞;CM-DIL;DAPI;荧光染料;标记;骨髓间充质干细胞;细胞活力;细胞增殖;心肌梗死;荧光表达;干细胞图片文章【作者】商青青;李凯;周建业;胡盛寿【作者单位】阜外心血管病医院，北京市 100037;阜外心血管病医院，北京市100037;阜外心血管病医院，北京市 100037;阜外心血管病医院，北京市 100037【正文语种】中文【中图分类】R394.20 引言 Introduction在有关细胞移植的实验研究中，细胞标记技术被用来追踪干细胞在宿主体内的去向和命运。
